Ribosome-inactivating proteins (RIPs) are plant proteins that are capable of catalytically inactivating eukaryotic ribosomes and are consequently extremely potent inhibitors of eukaryotic protein synthesis. RIPs have been divided into two classes: type 1 and type 2 RIPs (see Barbieri and Stirpe (1982), Cancer Surveys., 1:489-520). There is significant amino acid sequence homology between members of both type 1 and type 2 RIPs, and with the bacterial Shiga and Shiga-like toxins which also have the same mechanism of action (see Hovde et al. (1988), Proc. Natl. Acad. Sci. USA, 85:2568-2572).
Type 2 RIPs consist of two polypeptides; an RIP (or A-chain) which is covalently attached via a disulfide bond to a lectin-like protein (or B-chain). The B-chain binds to cell surface carbohydrates and facilitates subsequent cellular internalization of the RIP A-chain moiety, which results in rapid inactivation of protein synthesis and cell death. Type 2 RIPs are therefore extremely potent cytotoxins and animal poisons, the best studied example of which is ricin.
In contrast, type 1 RIPs characterized to date consist of a single polypeptide chain equivalent in activity to that of A-chain RIPs but lacking a covalently attached B-chain. Consequently, type 1 RIPs are scarcely toxic to intact cells but retain their extreme potency against cell-free protein translation systems. Typical IC.sub.50 concentrations of type 1 RIPs are 0.5 to 10 ng/ml (0.16 to 33 pM). Until the discoveries detailed hereinbelow, reported type 1 RIPs were a remarkably homogeneous class of basic proteins with Mr values of about 30,000. Type 1 RIPs are found in a great variety of dicot and monocot plants and they may be ubiquitous. They are often abundant proteins in seeds, roots or latex for example. Their in vivo function is unclear but it has been proposed that they may be antiviral agents (see Stevens et al. (1981), Experientia, 37:257-259) or antifungal agents (see Roberts and Seltrennikkoff (1986), Bioscience Reports, 6:19-29).
To date, one article has discussed the presence of an inhibitor of animal cell-free protein synthesis in maize, as well as other cereal crops (see Coleman and Roberts (1982), Biochimica et Biophysica Acta, 696:239-244). The preparation of the maize inhibitor was via ammonium sulfate precipitation and phosphocellulose column chromatography. It is generally believed that the inhibitor isolated from maize was pure. The reported molecular weight of the inhibitor was 23 kiloDaltons (kD), with a reported IC.sub.50 of 50 to 100 ng/ml in an ascites cell-free protein synthesis assay.
Where the effect of RIPs on ribosomes has been examined, both type 1 and type 2 RIPs possess a unique and highly specific N-glycosidase activity which cleaves the glycosidic bond of adenine 4324 of the rat liver ribosomal 28S RNA (see Endo (1988), In:Immunotoxins, Frankel (ed.), supra).
Commercial interest in RIPs has primarily focused on their use in construction of therapeutic toxins targeted to specific cells such as tumor cells by attachment of a targeting polypeptide, most frequently a monoclonal antibody (see Immunotoxins (1988), supra). This mimics the binding functionality of the B-chain of type 2 RIPs but replaces the non-specific action of B chains with a highly selective ligand. Another recent potential use is in HIV therapy (see U.S. Pat. No. 4,869,903 to Lifson et al., (Genelabs Incorporated and The Regents of the University of California)).
However, while a maize-derived protein synthesis inhibitor, like protein synthesis inhibitors from other Panicoideae, would appear to be useful for construction of cytotoxic conjugates, no artisan to date has reported to have successfully characterized a Panicoideae RIP. This is somewhat surprising in view of the success achieved with RIPs from non-Panicoideae plants, including cereals such as barley (see Lambert et al. (1988), In:Immunotoxins, supra). In part, the lack of success to date by skilled artisans in successfully utilizing the maize RIP described by Coleman and Roberts may be attributed to the fact that the protein synthesis inhibitor was relatively uncharacterized and reported IC.sub.50 was relatively poor.
There is interest in expressing recombinant RIP in commonly employed host eukaryotic cells, because of the capacity to provide correct post-translational processing. However, as RIPs effectively inhibit protein synthesis in eukaryotic cells, a predictable problem is that heterologous expression of an RIP will result in host cell death. Thus, eukaryotic cells are generally not used as recombinant host cells. Although eukaryotic cells may be used in certain circumstances, the RIP must be constructed so as to be secreted prior to the cell experiencing toxicity (see EP 0 335 476 to Gelfand et al. (Cetus Corp.)). Therefore, prokaryotic host cells are generally used as hosts, notwithstanding disadvantages such as the inability to glycosylate and properly fold heterologously-expressed proteins.
It is thus an object of the invention to provide a method of preparing inactive forms of RIPs, in which an inactive RIP may be expressed by eukaryotic host cells and then converted to an active form.
It is yet another object of the invention to provide the DNA sequence of a gene encoding at least one inactive form of RIP, as well as expression vehicles, host cells and cell cultures containing such DNA sequence.
Other objects and advantages of the present invention will become apparent from the teachings presented hereinafter.
It is to these objects to which the present invention is directed.